DNA extracted from 20 genotypes was digested with the restriction enzymes EcoRI and EcoRV and heterologous ctDNA sequences from barley and pea were used as probes to detect Rubus ctDNA sequences on Southern blots of Rubus total DNA. RFLP was detected and 92 restriction fragments were generated by the probe/enzyme combinations examined. Cladistic principles based on the parsimony assumption were used to assemble a phylogenetic tree based on chloroplast restriction fragment length data. The phylogenetic tree grouped the taxonomically defined species and was in general agreement with information based on morphological criteria. R. illecebrosus was shown to have diverged considerably in terms of evolutionary time from other species in subgenus Idaeobatus.